GBS bacteria are a recognized etiological agent for bacteremia and/or meningitis in infants, and for infections in adults. Baker, xe2x80x9cGroup B Streptococcal Infectionsxe2x80x9d in Advances in Internal Medicine, 25:475-500 (1980). Accordingly, it is important to develop rapid and definitive assays for diagnosis of GBS infection, and methods of generating protection against GBS, particularly in infants and compromised individuals.
The capsular polysaccharides from GBS bacteria are known to be important to GBS virulence and the development of protective immunity. See Kasper et al. U.S. Pat. No. 5,302,386. Moreover, the CP of recognized GBS types (I-V) are chemically related but antigenically distinct having repeating structures composed of galactose, glucose, N-acetyl glucosamine, and N-acetyl-neuraminic (sialic) acid.
Infants and young children have poor immunogenic response to polysaccharide antigens. These responses are characterized as being T cell independent and therefore are not associated with important attributes such as memory, isotype switching, or affinity maturation, which are necessary for conferring long term immunologic protection against subsequent infection. To circumvent this lack of an effective immunogenic response in infants and young children to polysaccharides, the art has developed means of converting the T cell independent response to T cell dependent response by covalently coupling polysaccharide bacterial antigens to a carrier protein to form a conjugate molecule. See, Jennings et al. U.S. Pat. No. 4,356,170, which is incorporated herein by reference.
Various procedures have been described in the art for conjugating capsular polysaccharides to proteins. For review, see Contributions to Microbiology and Immunology, vol 10, Conjugate Vaccines, volume editions J. M. Cruse and R. E. Lewis, Jr., 1989, which is incorporated herein by reference. In one method, polysaccharide is subjected to mild acid hydrolysis to produce reducing end groups capable of reacting with protein to form a covalent bond. Anderson, P. A., Infect. Immun., 39:233-238 (1983). However, the terminal sugar groups which participate in conjugating to protein exist in equilibrium between a hemiacetal and aldehyde and therefore couple to protein with poor efficiency. To overcome the poor reactivity of the terminal reducing sugar, the art turned to mild oxidation to introduce stable aldehyde groups at terminal positions of polysaccharides used to conjugate to protein. Jennings et al. U.S. Pat. No. 4,356,170, supra.
Kasper et al. U.S. Pat. No. 5,302,386 and International application WO 94/06467, respectively relate to GBS type III and II conjugate vaccines, both of which are incorporated herein by reference. According to the U.S. Pat. No. 5,302,386, endo-xcex2-galactosidase is used to cleave the polysaccharide backbone to produce products suitable for conjugating to protein. Oxidation of at least two terminal sialic acid groups to produce cross-linked conjugates is described in the WO 94/06467 application.
Type III GBS capsular polysaccharides are composed of a backbone of repeating branched pentasaccharide units. Jennings et al., Canadian J. Biochem., 58:112-120 (1980). One study of type III GBS polysaccharides reports that the natural immunodeterminant site is located at the side chain-backbone junction. Jennings et al., Biochemistry, 20:4511-4518 (1980). The presence of the side chain terminal N-acetyl-neuraminic acid residues reportedly was critical for immunodeterminant expression.
Prior methods of depolymerizing GBS II or III polysaccharides rely on either costly enzymatic methods or on acid hydrolysis which may alter the antigenicity of the CP due to removal of the labile terminal sialic acid groups. Accordingly, there is a need for relatively inexpensive and mild chemical procedures which are effective for depolymerizing GBS type II and III CP in a manner which results in fragments which are useful for producing CP-protein conjugate vaccines.
This invention relates to a method of depolymerizing Group B Streptococcus type II (GBS-II) and type III (GBS-III) capsular polysaccharides (CP) by deaminative cleavage to generate products terminating with a 2,5-anhydro-D-mannose structure. According to this invention, the GBS-II CP and GBS-III CP are treated with sodium hydroxide and a nitrosation reagent such as sodium nitrite to depolymerize the GBS polysaccharides to produce fragments having a terminal aldehyde group located at the end of the polysaccharide backbone. The resulting CP fragments are antigenic and are also useful for conjugating to protein to produce immunogens which are effective for eliciting protective immune responses in mammals including neonates.
Another embodiment of this invention therefore is a method of making a conjugate molecule for use as a vaccine. The method comprises subjecting GBS-II or GBS-III CP to treatment by base and a diazonium salt forming reagent to form a fragment terminating with a 2,5-anhydro-D-mannose residue. The 2,5-anhydro-D-mannose terminating fragment is then combined with a protein and subjected to reductive amination to form the conjugate molecules of the invention. Accordingly, another aspect of this invention are GBS-II and GBS-III CP conjugate molecules comprising GBS-II or GBS-III CP fragments linked to protein through a terminal 2,5-anhydro-D-mannose. Because the process of depolymerizing the GBS type II and type III polysaccharides generates fragments having a single reactive site at the terminal end of the backbone, this invention provides a means of producing conjugate molecules wherein each GBS type II or III polysaccharide chain is bound to a single protein, each by a secondary amine through the terminal reducing sugar.
The conjugates of this invention are useful as active vaccines for immunizing individuals against GBS-II and GBS-III bacterial infection. Also provided by this invention are multivalent vaccines comprising polysaccharides derived from different serotypes or species of bacteria.
In addition, this invention encompasses immune serum or antibodies raised in response to immunization with the conjugate molecules of this invention and which are useful as reagents for detecting the presence of GBS type II or III bacteria or as vaccines for conferring passive immunity.
Another embodiment of this invention are methods and compositions useful for separating and/or detecting GBS type II or type III antibodies. According to one mode of practicing this embodiment, the polysaccharide fragments prepared according to this invention are immobilized onto a solid support. By combining a source of antibody such as serum, with the polysaccharide fragment bound to the solid support, the antibody which bonds to the polysaccharide fragment may be detected by standard immunoassay techniques or separated from the starting material or serum.
An object of this invention is to provide methods for fragmenting GBS type II and III polysaccharides to produce fragments useful producing conjugate molecules. Another object of this invention is to produce GBS type II and type III polysaccharide molecules which are useful as vaccines to protect against infection and as immunoreagents.